The following discussion of the background is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art.
Prokaryotes have been widely used for the production of recombinant proteins. Controlled expression of the desired polypeptide or protein is accomplished by coupling the gene encoding the protein through recombinant DNA techniques behind a promoter, the activity of which can be regulated by external factors. This expression construct is carried on a vector, most often a plasmid. Introduction of the plasmid carrying the expression construct into a host bacterium and culturing that organism in the presence of compounds which activate the promoter results in expression of the desired protein. In this way, large quantities of the desired protein can be produced.
E. coli is the most commonly used prokaryote for protein production. A variety of plasmid vectors have been developed for use in E. coli, which employ several different types of promoters, selectable markers, and origins of replication. In the most common arrangement, the expressed protein accumulates in the cytoplasm. While this approach is useful for some proteins, not all proteins can be accumulated in the cytoplasm in an active state. Often, when the desired protein is produced at high levels, it may be toxic to the host cell, or accumulate as an insoluble particle known as an inclusion body. Proteins which accumulate as inclusion bodies are difficult to recover in an active form. In such cases, it may be desirable to engineer the protein so that it is secreted from the cell.
E. coli and other gram-negative bacteria are generally considered poor hosts for secreted protein production. There are no well-understood secretory pathways in E. coli to transport heterologous proteins to the extracellular environment. The recent discovery of YebF-mediated secretion (Nat Biotechnol. 2006. 24(1):100-4) is the first report of a native E. coli system capable of secreting both the native protein, YebF, and translational fusions to YebF. However, the expression level of YebF fusion proteins is typically low.